Concepts and tools for gene editing

2017 ◽  
Vol 29 (1) ◽  
pp. 1 ◽  
Author(s):  
Santiago Josa ◽  
Davide Seruggia ◽  
Almudena Fernández ◽  
Lluis Montoliu
Keyword(s):  
Dna Sequences ◽  
Gene Editing ◽  
Embryonic Stem ◽  
Zinc Finger Nucleases ◽  
Technological Advancement ◽  
Transcription Activator ◽  
Genetic Modifications ◽  
Associated Proteins ◽  
At Will ◽  
Cas Gene

Gene editing is a relatively recent concept in the molecular biology field. Traditional genetic modifications in animals relied on a classical toolbox that, aside from some technical improvements and additions, remained unchanged for many years. Classical methods involved direct delivery of DNA sequences into embryos or the use of embryonic stem cells for those few species (mice and rats) where it was possible to establish them. For livestock, the advent of somatic cell nuclear transfer platforms provided alternative, but technically challenging, approaches for the genetic alteration of loci at will. However, the entire landscape changed with the appearance of different classes of genome editors, from initial zinc finger nucleases, to transcription activator-like effector nucleases and, most recently, with the development of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas). Gene editing is currently achieved by CRISPR–Cas-mediated methods, and this technological advancement has boosted our capacity to generate almost any genetically altered animal that can be envisaged.

scholarly journals Gene editing and CRISPR in the clinic: current and future perspectives

2020 ◽  
Vol 40 (4) ◽  
Author(s):  
Matthew P. Hirakawa ◽  
Raga Krishnakumar ◽  
Jerilyn A. Timlin ◽  
James P. Carney ◽  
Kimberly S. Butler
Keyword(s):  
Clinical Trials ◽  
Genome Editing ◽  
Dna Sequences ◽  
Gene Editing ◽  
Ex Vivo ◽  
Scientific Basis ◽  
Current Status ◽  
Zinc Finger Nucleases ◽  
Transcription Activator

Abstract Genome editing technologies, particularly those based on zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR (clustered regularly interspaced short palindromic repeat DNA sequences)/Cas9 are rapidly progressing into clinical trials. Most clinical use of CRISPR to date has focused on ex vivo gene editing of cells followed by their re-introduction back into the patient. The ex vivo editing approach is highly effective for many disease states, including cancers and sickle cell disease, but ideally genome editing would also be applied to diseases which require cell modification in vivo. However, in vivo use of CRISPR technologies can be confounded by problems such as off-target editing, inefficient or off-target delivery, and stimulation of counterproductive immune responses. Current research addressing these issues may provide new opportunities for use of CRISPR in the clinical space. In this review, we examine the current status and scientific basis of clinical trials featuring ZFNs, TALENs, and CRISPR-based genome editing, the known limitations of CRISPR use in humans, and the rapidly developing CRISPR engineering space that should lay the groundwork for further translation to clinical application.

scholarly journals Principles of Genetic Engineering

Genes ◽  
2020 ◽  
Vol 11 (3) ◽  
pp. 291 ◽  
Author(s):  
Thomas M. Lanigan ◽  
Huira C. Kopera ◽  
Thomas L. Saunders
Keyword(s):  
Homologous Recombination ◽  
Genetic Engineering ◽  
Dna Sequences ◽  
Cultured Cells ◽  
Embryonic Stem ◽  
Zinc Finger Nucleases ◽  
Chromosome Break ◽  
Transcription Activator ◽  
Random Integration ◽  
Dna Insertion

Genetic engineering is the use of molecular biology technology to modify DNA sequence(s) in genomes, using a variety of approaches. For example, homologous recombination can be used to target specific sequences in mouse embryonic stem (ES) cell genomes or other cultured cells, but it is cumbersome, poorly efficient, and relies on drug positive/negative selection in cell culture for success. Other routinely applied methods include random integration of DNA after direct transfection (microinjection), transposon-mediated DNA insertion, or DNA insertion mediated by viral vectors for the production of transgenic mice and rats. Random integration of DNA occurs more frequently than homologous recombination, but has numerous drawbacks, despite its efficiency. The most elegant and effective method is technology based on guided endonucleases, because these can target specific DNA sequences. Since the advent of clustered regularly interspaced short palindromic repeats or CRISPR/Cas9 technology, endonuclease-mediated gene targeting has become the most widely applied method to engineer genomes, supplanting the use of zinc finger nucleases, transcription activator-like effector nucleases, and meganucleases. Future improvements in CRISPR/Cas9 gene editing may be achieved by increasing the efficiency of homology-directed repair. Here, we describe principles of genetic engineering and detail: (1) how common elements of current technologies include the need for a chromosome break to occur, (2) the use of specific and sensitive genotyping assays to detect altered genomes, and (3) delivery modalities that impact characterization of gene modifications. In summary, while some principles of genetic engineering remain steadfast, others change as technologies are ever-evolving and continue to revolutionize research in many fields.

Evaluating and Enhancing Target Specificity of Gene-Editing Nucleases and Deaminases

2019 ◽  
Vol 88 (1) ◽  
pp. 191-220 ◽  
Author(s):  
Daesik Kim ◽  
Kevin Luk ◽  
Scot A. Wolfe ◽  
Jin-Soo Kim
Keyword(s):  
Dna Sequences ◽  
Gene Editing ◽  
Genomic Rearrangements ◽  
Zinc Finger Nucleases ◽  
Collateral Damage ◽  
Transcription Activator ◽  
Genome Wide ◽  
Target Sites ◽  
Programmable Nucleases ◽  
Target Activity

Programmable nucleases and deaminases, which include zinc-finger nucleases, transcription activator-like effector nucleases, CRISPR RNA-guided nucleases, and RNA-guided base editors, are now widely employed for the targeted modification of genomes in cells and organisms. These gene-editing tools hold tremendous promise for therapeutic applications. Importantly, these nucleases and deaminases may display off-target activity through the recognition of near-cognate DNA sequences to their target sites, resulting in collateral damage to the genome in the form of local mutagenesis or genomic rearrangements. For therapeutic genome-editing applications with these classes of programmable enzymes, it is essential to measure and limit genome-wide off-target activity. Herein, we discuss the key determinants of off-target activity for these systems. We describe various cell-based and cell-free methods for identifying genome-wide off-target sites and diverse strategies that have been developed for reducing the off-target activity of programmable gene-editing enzymes.

scholarly journals Delivery Approaches for Therapeutic Genome Editing and Challenges

Genes ◽  
2020 ◽  
Vol 11 (10) ◽  
pp. 1113 ◽  
Author(s):  
Ilayda Ates ◽  
Tanner Rathbone ◽  
Callie Stuart ◽  
P. Hudson Bridges ◽  
Renee N. Cottle
Keyword(s):  
Clinical Trials ◽  
Genome Editing ◽  
Zinc Finger ◽  
Gene Editing ◽  
Zinc Finger Nucleases ◽  
Systemic Delivery ◽  
Transcription Activator ◽  
Therapeutic Gene ◽  
Major Barrier ◽  
Associated Proteins

Impressive therapeutic advances have been possible through the advent of zinc-finger nucleases and transcription activator-like effector nucleases. However, discovery of the more efficient and highly tailorable clustered regularly interspaced short palindromic repeats (CRISPR) and associated proteins (Cas9) has provided unprecedented gene-editing capabilities for treatment of various inherited and acquired diseases. Despite recent clinical trials, a major barrier for therapeutic gene editing is the absence of safe and effective methods for local and systemic delivery of gene-editing reagents. In this review, we elaborate on the challenges and provide practical considerations for improving gene editing. Specifically, we highlight issues associated with delivery of gene-editing tools into clinically relevant cells.

scholarly journals Exploiting DNA Endonucleases to Advance Mechanisms of DNA Repair

Biology ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 530
Author(s):  
Marlo K. Thompson ◽  
Robert W. Sobol ◽  
Aishwarya Prakash
Keyword(s):  
Dna Repair ◽  
Mammalian Cells ◽  
Genome Stability ◽  
Gene Editing ◽  
Adaptive Immune System ◽  
Zinc Finger Nucleases ◽  
Specific Gene ◽  
Target Sequence ◽  
Transcription Activator ◽  
Low Cytotoxicity

The earliest methods of genome editing, such as zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs), utilize customizable DNA-binding motifs to target the genome at specific loci. While these approaches provided sequence-specific gene-editing capacity, the laborious process of designing and synthesizing recombinant nucleases to recognize a specific target sequence, combined with limited target choices and poor editing efficiency, ultimately minimized the broad utility of these systems. The discovery of clustered regularly interspaced short palindromic repeat sequences (CRISPR) in Escherichia coli dates to 1987, yet it was another 20 years before CRISPR and the CRISPR-associated (Cas) proteins were identified as part of the microbial adaptive immune system, by targeting phage DNA, to fight bacteriophage reinfection. By 2013, CRISPR/Cas9 systems had been engineered to allow gene editing in mammalian cells. The ease of design, low cytotoxicity, and increased efficiency have made CRISPR/Cas9 and its related systems the designer nucleases of choice for many. In this review, we discuss the various CRISPR systems and their broad utility in genome manipulation. We will explore how CRISPR-controlled modifications have advanced our understanding of the mechanisms of genome stability, using the modulation of DNA repair genes as examples.

Gene editing as a promising approach for respiratory diseases

2018 ◽  
Vol 55 (3) ◽  
pp. 143-149 ◽  
Author(s):  
Yichun Bai ◽  
Yang Liu ◽  
Zhenlei Su ◽  
Yana Ma ◽  
Chonghua Ren ◽  
...  
Keyword(s):  
Respiratory Diseases ◽  
Gene Editing ◽  
Gene Mutations ◽  
Well Being ◽  
Short Review ◽  
Zinc Finger Nucleases ◽  
Transcription Activator ◽  
Mortality And Morbidity ◽  
Chronic Respiratory Diseases ◽  
Difficulty Breathing

Respiratory diseases, which are leading causes of mortality and morbidity in the world, are dysfunctions of the nasopharynx, the trachea, the bronchus, the lung and the pleural cavity. Symptoms of chronic respiratory diseases, such as cough, sneezing and difficulty breathing, may seriously affect the productivity, sleep quality and physical and mental well-being of patients, and patients with acute respiratory diseases may have difficulty breathing, anoxia and even life-threatening respiratory failure. Respiratory diseases are generally heterogeneous, with multifaceted causes including smoking, ageing, air pollution, infection and gene mutations. Clinically, a single pulmonary disease can exhibit more than one phenotype or coexist with multiple organ disorders. To correct abnormal function or repair injured respiratory tissues, one of the most promising techniques is to correct mutated genes by gene editing, as some gene mutations have been clearly demonstrated to be associated with genetic or heterogeneous respiratory diseases. Zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) systems are three innovative gene editing technologies developed recently. In this short review, we have summarised the structure and operating principles of the ZFNs, TALENs and CRISPR/Cas9 systems and their preclinical and clinical applications in respiratory diseases.

scholarly journals The Role of Gene Editing in Neurodegenerative Diseases

2018 ◽  
Vol 27 (3) ◽  
pp. 364-378 ◽  
Author(s):  
Hueng-Chuen Fan ◽  
Ching-Shiang Chi ◽  
Yih-Jing Lee ◽  
Jeng-Dau Tsai ◽  
Shinn-Zong Lin ◽  
...  
Keyword(s):  
Neurodegenerative Diseases ◽  
Gene Editing ◽  
Clinical Manifestations ◽  
Zinc Finger Nucleases ◽  
Diagnostic Tools ◽  
Pathological Feature ◽  
Transcription Activator ◽  
Substantial Impact ◽  
Parkinson’S Diseases ◽  
The Impact

Neurodegenerative diseases (NDs), at least including Alzheimer’s, Huntington’s, and Parkinson’s diseases, have become the most dreaded maladies because there are no precise diagnostic tools or definite treatments for these debilitating diseases. The increased prevalence and a substantial impact on the social–economic and medical care of NDs propel governments to develop policies to counteract the impact. Although the etiologies of NDs are still unknown, growing evidence suggests that genetic, cellular, and circuit alternations may cause the generation of abnormal misfolded proteins, which uncontrolledly accumulate to damage and eventually overwhelm the protein-disposal mechanisms of these neurons, leading to a common pathological feature of NDs. If the functions and the connectivity can be restored, alterations and accumulated damages may improve. The gene-editing tools including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats–associated nucleases (CRISPR/CAS) have emerged as a novel tool not only for generating specific ND animal models for interrogating the mechanisms and screening potential drugs against NDs but also for the editing sequence-specific genes to help patients with NDs to regain function and connectivity. This review introduces the clinical manifestations of three distinct NDs and the applications of the gene-editing technology on these debilitating diseases.

scholarly journals Genome editing for blood disorders: state of the art and recent advances

2019 ◽  
Vol 3 (3) ◽  
pp. 289-299 ◽  
Author(s):  
Marianna Romito ◽  
Rajeev Rai ◽  
Adrian J. Thrasher ◽  
Alessia Cavazza
Keyword(s):  
Gene Editing ◽  
Therapeutic Potential ◽  
State Of The Art ◽  
Clinical Success ◽  
Zinc Finger Nucleases ◽  
Transcription Activator ◽  
Blood Disorders ◽  
Flexible Tool ◽  
Wide Range ◽  
Made In

Abstract In recent years, tremendous advances have been made in the use of gene editing to precisely engineer the genome. This technology relies on the activity of a wide range of nuclease platforms — such as zinc-finger nucleases, transcription activator-like effector nucleases, and the CRISPR–Cas system — that can cleave and repair specific DNA regions, providing a unique and flexible tool to study gene function and correct disease-causing mutations. Preclinical studies using gene editing to tackle genetic and infectious diseases have highlighted the therapeutic potential of this technology. This review summarizes the progresses made towards the development of gene editing tools for the treatment of haematological disorders and the hurdles that need to be overcome to achieve clinical success.

scholarly journals Applications of CRISPR/Cas Gene-Editing Technology in Fungi

2021 ◽  
Author(s):  
Binyou Liao ◽  
Lei Cheng ◽  
Yujie Zhou ◽  
Yangyang Shi ◽  
Xingchen Ye ◽  
...  
Keyword(s):  
Gene Editing ◽  
Yeast Genome ◽  
Transcription Activator ◽  
Single Nucleotide ◽  
Complex Design ◽  
Infection Treatment ◽  
New Gene ◽  
Low Efficiency ◽  
Genomic Editing ◽  
Cas Gene

Abstract Genome editing technology develop fast in recent years. The traditional gene-editing methods, including homologous recombination, zinc finger endonuclease, and transcription activator-like effector nuclease and so on, which have greatly promoted the research of genetics and molecular biology, have gradually showed their limitations such as low efficiency, high error rate, and complex design. In 2012,a new gene-editing technology, the CRISPR/Cas9 system, was setup based on the research of the immune responses to viruses from archaea and bacteria. Due to its advantages of high target efficiency, simple primer design, and wide application, CRISPR/Cas9 system, whose developers are awared the Nobel Prize in Chemistry this year, has become the dominant genomic editing technology in global academia and some pharmaceuticals. Here we briefly introduce the CRISPR/Cas system and its main applications in yeast, filamentous fungi and macrofungi, including single nucleotide, polygene and polyploid editing, yeast chromosome construction, yeast genome and yeast library construction, CRISPRa/CRISPRi-mediated, CRISPR platform of non-traditional yeast and regulation of metabolic pathway, to highlight the possible applications on fungal infection treatment and to promote the transformation and application of the CRISPR/Cas system in fungi.

scholarly journals Update of Regulatory Options of New Breeding Techniques and Biosafety Approaches among Selected Countries: A Review

2020 ◽  
pp. 18-35
Author(s):  
Silas Obukosia ◽  
Olalekan Akinbo ◽  
Woldeyesus Sinebo ◽  
Moussa Savadogo ◽  
Samuel Timpo ◽  
...  
Keyword(s):  
Genome Editing ◽  
Genetically Modified Organisms ◽  
Zinc Finger Nucleases ◽  
Intermediate Step ◽  
Homing Endonucleases ◽  
Transcription Activator ◽  
Associated Proteins ◽  
Genomic Editing ◽  
New Breeding Techniques ◽  
Breeding Techniques

A new set of breeding techniques, referred to as New Breeding Techniques developed in the last two decades have potential for enhancing improved productivity in crop and animal breeding globally. These include site directed nucleases based genomic editing procedures-CRISPR and Cas associated proteins, Zinc Finger Nucleases, Meganucleases/Homing Endonucleases and Transcription- Activator Like-Effector Nucleases for genome editing and other technologies including- Oligonucleotide-Directed Mutagenesis, Cisgenesis and intragenesis, RNA-Dependent DNA methylation; Transgrafting, Agroinfiltration, Reverse breeding. There are ongoing global debates on whether the processes of and products emerging from these technologies should be regulated as genetically modified organisms or approved as conventional products. Decisions on whether to regulate as GMOs are based both on understanding of the molecular basis of their development and if the GMO intermediate step was used. For example- cisgenesis, can be developed using Agrobacterium tumefaciens methods of transformation, a process used by GMO but if the selection is properly conducted the intermediate GMO elements will be eliminated and the final product will be identical to the conventionally developed crops. Others like Site Directed Nuclease 3 are regulated as GMOs in countries such as United State of America, Canada, European Union, Argentina, Australia. Progress in genome editing research, testing of genome edited bacterial blight resistant rice, development of Guidelines for regulating new breeding techniques or genome editing in Africa is also covered with special reference to South Africa, Kenya and Nigeria. Science- and evidence-based approach to regulation of new breeding techniques among regulators and policy makers should be strongly supported.

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